A Review of Advances in Molecular Imaging of Rheumatoid Arthritis: From In Vitro to Clinic Applications Using Radiolabeled Targeting Vectors with Technetium-99m

Rheumatoid arthritis (RA) is a systemic autoimmune disorder caused by inflammation of cartilaginous diarthrodial joints that destroys joints and cartilage, resulting in synovitis and pannus formation. Timely detection and effective management of RA are pivotal for mitigating inflammatory arthritis consequences, potentially influencing disease progression. Nuclear medicine using radiolabeled targeted vectors presents a promising avenue for RA diagnosis and response to treatment assessment. Radiopharmaceutical such as technetium-99m (99mTc), combined with single photon emission computed tomography (SPECT) combined with CT (SPECT/CT), introduces a more refined diagnostic approach, enhancing accuracy through precise anatomical localization, representing a notable advancement in hybrid molecular imaging for RA evaluation. This comprehensive review discusses existing research, encompassing in vitro, in vivo, and clinical studies to explore the application of 99mTc radiolabeled targeting vectors with SPECT imaging for RA diagnosis. The purpose of this review is to highlight the potential of this strategy to enhance patient outcomes by improving the early detection and management of RA.


Introduction
Rheumatoid arthritis (RA) is a persistent, systemic, inflammatory autoimmune disease that primarily affects the synovial joints, causing damage to cartilage and bone and extraarticular involvement such as the skin, eyes, lungs, digestive system, nervous system, heart, and kidney [1,2].Common symptoms of RA include morning stiffness lasting for more than 30 min in the affected joint accompanied by swelling, appetite loss and a limited range of motion [3].Approximately 1% of the global population suffers from RA [4], and women have a threefold higher likelihood of developing RA than men [5][6][7].The annual expenses of RA are estimated to be $48 billion (2016 US dollars) in United states.These costs include direct healthcare costs as well as indirect costs such as lost productivity and intangible costs to the employer and family [8,9].According to a recent modeling analysis, the yearly economic burden of RA in Italy was projected to be approximately EUR 2.0 billion in 2015.Direct medical expenses accounted for 45% of the expenditure.Indirect costs accounted for nearly half of the expenditure, around 45%, while direct non-medical expenditures determined the remaining 10% [10].
Indirect costs accounted for nearly half of the expenditure, around 45%, while direct non medical expenditures determined the remaining 10% [10].

Molecular Imaging
In molecular imaging, biological processes in living systems can be observed, analyzed, and measured at the molecular by imaging modalities such as ultrasound, magnetic resonance imaging (MRI), positron emission tomography (PET), and single photon emission computed tomography (SPECT) [53,54,55,56,57].PET and SPECT are sophisticated imaging modalities utilized to visualize biological processes at the molecular level [58,59,60,61].PET is more sensitive while SPECT is more widely available, less expensive, and has better imaging properties than PET [62].The estimated cost of SPECT/CT imaging in a clinical setting is around $622 per patient per year [63].
In SPECT imaging, gamma rays emitted by radionuclides are detected using a gamma camera, which is linked with a computer algorithm to transform radioactivity emitted from radionuclides (possessing diagnostic information) into a precise picture of the distribution of radionuclides in the body [58,59,60,64,65].Most radiopharmaceuticals

Molecular Imaging
In molecular imaging, biological processes in living systems can be observed, analyzed, and measured at the molecular by imaging modalities such as ultrasound, magnetic resonance imaging (MRI), positron emission tomography (PET), and single photon emission computed tomography (SPECT) [53][54][55][56][57]. PET and SPECT are sophisticated imaging modalities utilized to visualize biological processes at the molecular level [58][59][60][61].PET is more sensitive while SPECT is more widely available, less expensive, and has better imaging properties than PET [62].The estimated cost of SPECT/CT imaging in a clinical setting is around $622 per patient per year [63].
In SPECT imaging, gamma rays emitted by radionuclides are detected using a gamma camera, which is linked with a computer algorithm to transform radioactivity emitted from radionuclides (possessing diagnostic information) into a precise picture of the distribution of radionuclides in the body [58][59][60]64,65].Most radiopharmaceuticals used in SPECT are single molecules radiolabeled with a gamma-emitting isotope, such as iodine-123, gallium-67, indium-111 or technetium-99m.SPECT provides 3D images that are more sensitive than structural images [66,67].The radiopharmaceutical agent is usually made of two elements, a carrier or ligand and a radioactive atom, which determines the diagnostic character of the radioactive compound through its nuclear properties.The carrier is crucial for precisely delivering the radionuclide to a biological target [68,69].Chelators and linkers are used to link ligands and radionuclides; indeed, radionuclides such as 99m Tc and 66/68 Ga require chelators for stability, while linkers are used optionally to increase radionuclides pharmacokinetics and binding affinity [70,71].
Clinical research radiotracers should meet the following criteria: short half-life, 100-600 keV monochromatic gamma ray emission for diagnostic applications; low toxicity, high stability, and high specificity; low plasma protein binding affinity; and rapid elimination [62,72,73].

Technetium-99m Overview
The radionuclide isotope technetium-99m ( 99m Tc) has been widely utilized in imaging and diagnostic procedures [74,75].99m Tc is the FDA-and EMA-approved and most common radioactive isotope tracer used in SPECT for imaging of the kidneys, brain, thyroid, heart, liver, gallbladder, spleen, salivary and lachrymal glands, sentinel nodes, and bone marrow [76].The short half-life of 99mTc makes it an ideal radionuclide for radiation exposure [74].The radioisotope technetium-99m emits gamma radiation (140.5 keV) and can be detected noninvasively on the outside of the body using imaging SPECT systems [77].
Technetium-99m can be introduced into bioactive molecules using inorganic technetium functional groups, which are synthesized in a physiological solution with a decreased oxidation state of technetium and labile coordination sites that are easily exploited to incorporate the desired bioactive component [65].
When combined with a suitable set of coordinating atoms, the metal fragments shown in Figure 3 offer a valuable method for attaching a physiological moiety to a technetium-99m [65].The radioactive metal fragment and a chelating group are attached to the bioactive molecule via a spacer group.Due to the strong affinity of the precursor metal fragment for particular binding sites on the ligand, two properly chosen molecular building blocks can form a conjugate complex [78].
the diagnostic character of the radioactive compound through its nuclear p carrier is crucial for precisely delivering the radionuclide to a biological Chelators and linkers are used to link ligands and radionuclides; indeed, such as 99m Tc and 66/68 Ga require chelators for stability, while linkers are used increase radionuclides pharmacokinetics and binding affinity [70,71].
Clinical research radiotracers should meet the following criteria: short 600 keV monochromatic gamma ray emission for diagnostic applications high stability, and high specificity; low plasma protein binding affinity; an nation [62,72,73].

Technetium-99m Overview
The radionuclide isotope technetium-99m ( 99m Tc) has been widely utiliz and diagnostic procedures [74,75].99m Tc is the FDA-and EMA-approved a mon radioactive isotope tracer used in SPECT for imaging of the kidneys, b heart, liver, gallbladder, spleen, salivary and lachrymal glands, sentinel no marrow [76].The short half-life of 99mTc makes it an ideal radionuclide for posure [74].The radioisotope technetium-99m emits gamma radiation (140.5 be detected noninvasively on the outside of the body using imaging SPECT Technetium-99m can be introduced into bioactive molecules using ino tium functional groups, which are synthesized in a physiological solutio creased oxidation state of technetium and labile coordination sites that are ea to incorporate the desired bioactive component [65].
When combined with a suitable set of coordinating atoms, the me shown in Figure 3 offer a valuable method for attaching a physiological mo netium-99m [65].The radioactive metal fragment and a chelating group a the bioactive molecule via a spacer group.Due to the strong affinity of the pr fragment for particular binding sites on the ligand, two properly chosen mo ing blocks can form a conjugate complex [78].

Potential Targets for the Molecular Imaging of Rheumatoid Arthritis
99mTc-labeled diphophonates can be used to visualize active synoviti and detect areas of increased bone activity potentially associated with arti action/destruction (Figure 4) [80].

Potential Targets for the Molecular Imaging of Rheumatoid Arthritis
99mTc-labeled diphophonates can be used to visualize active synovitis in RA joints and detect areas of increased bone activity potentially associated with articular bone reaction/destruction (Figure 4) [80].
However, a three-phase bone scan with 99m Tc-diphosphonates is suboptimal for predicting active inflammation in RA [81,82].99m Tc-diphosphonates can be used to observe postsurgical complications, fractures, or orthopedic hardware complications [83,84].

Monoclonal Antibodies
In 2006, rituximab was approved for the treatment of RA [93,94].Rituximab is a genetically engineered monoclonal antibody to CD20 antigen [95].CD20 antigen is expressed on B lymphocytes [96].B lymphocytes play an important role in RA pathogenesis.B lymphocytes release proinflammatory cytokines, ACPAs and rheumatoid factors (RFs) [97].B lymphocytes also express costimulatory molecules which facilitate T-cell activation [98], which involves stimulating and activating fibroblasts and macrophages [99][100][101][102].Rituximab binds to the CD antigen, disrupts signaling pathways, and triggers apoptosis via various mechanisms, such as Antibody-dependent cellular cytotoxicity (ADCC) and Complement-dependent cytotoxicity (CDC).In one study, 20 patients with various chronic inflammatory autoimmune disorders received rituximab, labeled with 99m Tc.At 6 and 20 h post-injury, whole-body scintigraphic images were acquired.Rapid absorption by the spleen and renal excretion of radioactivity was observed by [ 99m Tc]Tc-rituximab scintigraphy.Patients with rheumatoid arthritis who had inflamed joints at 6 h post-infection displayed varying degrees of uptake.These findings demonstrated that in individuals with autoimmune illnesses, this method can be used to evaluate B lymphocyte infiltration into impacted organs.The RAJI cell line was used to determine the binding affinity of [99mTc]Tc-rituximab to CD20 receptors.The binding affinity was greater than that of native rituximab, which has a Kd of 5.2 nM.This finding implies that rituximab maintained the ability to selectively bind to CD20 receptors even after technetium-99m radiolabeling.(Figure 5) [103].

Monoclonal Antibodies
In 2006, rituximab was approved for the treatment of RA [93,94].Rituximab is a genetically engineered monoclonal antibody to CD20 antigen [95].CD20 antigen is expressed on B lymphocytes [96].B lymphocytes play an important role in RA pathogenesis.B lymphocytes release proinflammatory cytokines, ACPAs and rheumatoid factors (RFs) [97].B lymphocytes also express costimulatory molecules which facilitate T-cell activation [98], which involves stimulating and activating fibroblasts and macrophages [99][100][101][102].Rituximab binds to the CD antigen, disrupts signaling pathways, and triggers apoptosis via various mechanisms, such as Antibody-dependent cellular cytotoxicity (ADCC) and Complement-dependent cytotoxicity (CDC).In one study, 20 patients with various chronic inflammatory autoimmune disorders received rituximab, labeled with 99m Tc.At 6 and 20 h post-injury, whole-body scintigraphic images were acquired.Rapid absorption by the spleen and renal excretion of radioactivity was observed by [ 99m Tc]Tc-rituximab scintigraphy.Patients with rheumatoid arthritis who had inflamed joints at 6 h post-infection displayed varying degrees of uptake.These findings demonstrated that in individuals with autoimmune illnesses, this method can be used to evaluate B lymphocyte infiltration into impacted organs.The RAJI cell line was used to determine the binding affinity of [99mTc]Tc-rituximab to CD20 receptors.The binding affinity was greater than that of native rituximab, which has a Kd of 5.2 nM.This finding implies that rituximab maintained the ability to selectively bind to CD20 receptors even after technetium-99m radiolabeling.(Figure 5) [103].However, a three-phase bone scan with 99m Tc-diphosphonates is suboptimal for predicting active inflammation in RA [81,82].99m Tc-diphosphonates can be used to observe postsurgical complications, fractures, or orthopedic hardware complications [83,84].

Monoclonal Antibodies
In 2006, rituximab was approved for the treatment of RA [93,94].Rituximab is a genetically engineered monoclonal antibody to CD20 antigen [95].CD20 antigen is expressed on B lymphocytes [96].B lymphocytes play an important role in RA pathogenesis.B lymphocytes release proinflammatory cytokines, ACPAs and rheumatoid factors (RFs) [97].B lymphocytes also express costimulatory molecules which facilitate T-cell activation [98], which involves stimulating and activating fibroblasts and macrophages [99][100][101][102].Rituximab binds to the CD antigen, disrupts signaling pathways, and triggers apoptosis via various mechanisms, such as Antibody-dependent cellular cytotoxicity (ADCC) and Complement-dependent cytotoxicity (CDC).In one study, 20 patients with various chronic inflammatory autoimmune disorders received rituximab, labeled with 99m Tc.At 6 and 20 h post-injury, whole-body scintigraphic images were acquired.Rapid absorption by the spleen and renal excretion of radioactivity was observed by [ 99m Tc]Tc-rituximab scintigraphy.Patients with rheumatoid arthritis who had inflamed joints at 6 h post-infection displayed varying degrees of uptake.These findings demonstrated that in individuals with autoimmune illnesses, this method can be used to evaluate B lymphocyte infiltration into impacted organs.The RAJI cell line was used to determine the binding affinity of [99mTc]Tc-rituximab to CD20 receptors.The binding affinity was greater than that of native rituximab, which has a Kd of 5.2 nM.This finding implies that rituximab maintained the ability to selectively bind to CD20 receptors even after technetium-99m radiolabeling.(Figure 5) [103].In another study, T-cell traffic and lymphocytic infiltration of organs affected by autoimmune illness were imaged using a radiolabeled anti-CD3 antibody.Visilizumab, [ 99m Tc]Tc-Life 2024, 14, 751 6 of 26 succinimidyl-6-hydrazinonicotinate hydrochloride (SHNH), was evaluated against HuT78 cells and BALB/c xenografted mice.The findings showed that HYNIC maintained its biochemical integrity and in vitro binding activity to CD3-positive cells while exhibiting high specific activity (SA; 10,360-11,100 MBq/mg) and labeling efficiency (>90%).The in vivo targeting experiment revealed that visilizumab labeled with 99mTc can be used for T lymphocyte trafficking and lymphocytic infiltration of tissues and organs [104].
In one study, it was determined whether administering tilmanocept subcutaneously (SC) with a 99m Tc label could identify macrophage-mediated inflammation in RA patients but not in healthy control (HC) subjects.[ 99m Tc]Tc-tilmanocept was injected into 18 RA patients.Patients were imaged via a whole-body planar scan at 2-3 h and 4-6 h post-injection.After the whole-body scan, 5 min planar images of both hands were obtained.The results revealed that [ 99m Tc]Tc-tilmanocept was significantly taken up by the affected joint [114].
Kardan et al. highlighted the potential use of intravenous administration of [ 99m Tc]Tctilmanocept in monitoring RA progression in the first-in-human phase I/phase II clinical study.The study involved 33 patients with active RA and six healthy volunteers, with various dose combinations of 99m Tc administered intravenously.The imaging results revealed that [ 99m Tc]Tc-tilmanocept can specifically localize inflamed joints, offering a foundation for a noninvasive method to monitor disease activity in macrophage-driven inflamed joints with RA [115].
The mitochondrial membrane translocator protein (TSPO) present on the macrophage surface can detect and quantify RA.In one study, 99m Tc-labeled DTPA-CB86 (Figure 6) was used to analyze TSPO binding affinity in RAW264.7 cells.Figure 7A shows the [ 99m Tc]Tc-DTPA-CB86 cell uptake ratio.In RAW264.7 cells, the peak accumulation of [ 99m Tc]Tc-DTPA-CB86 occurred 180 min later, reaching 36.45 ± 2.18% of the applied activity.A receptor saturation test confirmed the binding of [ 99m Tc]Tc-DTPA-CB86 to TSPO, revealing an IC50 value of 0.49 nM (Figure 7B).Moreover, cell efflux tests demonstrated that [ 99m Tc]Tc-DTPA-CB86 displayed robust cell retention in the cell line, with approximately 13.99% efflux observed after 4.5 to 8 h of incubation (Figure 7C).These findings suggest that the labeling procedure did no affect the ability of CB86 to bind selectively to TSPO.[ 99m Tc]Tc-DTPA-CB86 rapidly accumulated in the inflamed ankle (Figure 8).After 180 min of administration, [ 99m Tc]Tc-DTPA-CB86 uptake in the inflamed ankle was 2.35 ± 0.10%, which was significantly increased than that in normal tissues using SPECT [116].
In another study, 99m Tc-labeled mannosylated dextran or [ 99m Tc]Tc-(CO)3-DCM20 was used to target MMRs for early detection of RA in a mouse model of collagen induced arthritis (CIA).2-Deoxy-2-18F-fluoro-D-glucose ([ 18 F]FDG) was used as a control.The results revealed that [ 99m Tc]Tc-(CO)3-DCM20 accumulated more strongly in the hindpaws, forepaws, and knee joints of CIA mice than in control mice.The radioactivity level of [ 99m Tc]Tc-(CO)3-DCM20 was significantly correlated with the paw clinical score [117].
Put et al. used the radiolabeled MMR-targeting nanobodies to detect CD11b+F4/80+ macrophages in the inflamed joints of mice (Figure 9).This observation provided a means of objectively measuring inflammation and further understanding arthritis pathophysiology [53].Put et al. used the radiolabeled MMR-targeting nanobodies to detect CD11b+F4/80+ macrophages in the inflamed joints of mice (Figure 9).This observation provided a means of objectively measuring inflammation and further understanding arthritis pathophysiology [53].Put et al. used the radiolabeled MMR-targeting nanobodies to detect CD11b+F4/80+ macrophages in the inflamed joints of mice (Figure 9).This observation provided a means of objectively measuring inflammation and further understanding arthritis pathophysiology [53].Put et al. used the radiolabeled MMR-targeting nanobodies to detect CD11b+F4/80+ macrophages in the inflamed joints of mice (Figure 9).This observation provided a means of objectively measuring inflammation and further understanding arthritis pathophysiology [53].

Vascular Endothelial Growth Factor
Vascular endothelial growth factor (VEGF) is a member of the mammalian peptide family [118].These glycoproteins can generate dimeric forms by creating disulfide bridges between two monomers using a particular sequence of cysteines [119].Each VEGF family

Vascular Endothelial Growth Factor
Vascular endothelial growth factor (VEGF) is a member of the mammalian peptide family [118].These glycoproteins can generate dimeric forms by creating disulfide bridges between two monomers using a particular sequence of cysteines [119].Each VEGF family member is a glycosylated peptide monomer that must undergo homodimerization or heterodimerization to activate its biological activity [120].The best-studied member of the VEGF family, VEGF-A (also known as VEGF), is found in several isoforms (e.g., VEGF-A121, VEGF-A145, VEGF-A165, VEGF-A183, VEGF-A189, and VEGF-A206) as a result of alternative splicing of mRNAs produced during the transcription of the human gene VEGFA [121][122][123].
Activated platelets, fibroblasts, lymphocytes, and macrophages also produce VEGF-A glycoproteins [124,125].Hypoxia is the primary factor that triggers the transcription of the mRNA that encodes VEGF-A [68,[126][127][128].In contrast to hypoxia, normal conditions rigorously control the concentration of HIF-1 in cells.Several hormones, particular growth factors, and cytokines (including interleukin 1b, IL-1b, and tumor necrosis factor alpha [TNF]), among others, are also crucial cellular stimulators of VEGF-A [129].
Levashova et al. tested whether the labeled form of VEGF could be used to image VEGF receptors in a mouse model [130].Mice were administered turpentine to induce thigh inflammation.After a few days, the mice were treated with Tc-hydroxynonicotinic acid-single-chain Cys-tagged vascular endothelial growth factor ([ 99m Tc]Tc-inVEGF) and examined via SPECT imaging (Figure 10).SPECT imaging revealed high [ 99m Tc]Tc-inVEGF uptake in the thigh region.It was concluded that the high uptake of scVEGF is linked to the increased expression of VEGFR-2, which is involved in angiogenesis [130].

Integrins
Integrins are heterodimers, glycoprotein transmembrane receptors consisting of the α and β subunits with binding sites for the Extracellular matrix (ECM) [132,133].Upon binding to the ECM, integrins remodel the ECM by upregulating the extrusion of various proteases [134].Integrins regulate various cellular functions, such as motility, survival,

Integrins
Integrins are heterodimers, glycoprotein transmembrane receptors consisting of the α and β subunits with binding sites for the Extracellular matrix (ECM) [132,133].Upon binding to the ECM, integrins remodel the ECM by upregulating the extrusion of various proteases [134].Integrins regulate various cellular functions, such as motility, survival, invasion, and inflammation [135][136][137][138][139]. The binding of αvβ3 and α5β1 to Arg-Gly-Asp (RGD) integrins plays an essential role in RA pathogenesis [140][141][142][143]. Their role in angiogenesis is to facilitate EC migration and survival [144].A cell recognition pattern known as the RGD sequence is present in ECM proteins, such as vitronectin, fibrinogen, and fibronectin [145].These proteins are required for interactions with integrins.αvβ3 and α5β are known to be as fibronectin receptors.Moreover, αvβ3 binds to osteo-pontin, fibronectin, bone sialoprotein, and vitronectin (Figure 11) [89].The discovery of the RGD motif has led to the development of numerous peptidic and nonpeptidic integrin ligands with varying degrees of specificity.Integrins exhibit distinct responses when interacting with different RGD-containing ECM proteins, recognizing them individually.Using RGD-based ligands for noninvasive molecular imaging assessment is useful for studying angiogenesis in RA patients [146].[ 99m Tc]Tc-RGD binding affinity was evaluated in a study using a single HUVEC cell line.αVβ3 integrins were antagonistically treated with radiolabeled RGD peptides, with RGE (Arg-Gly-Glu) serving as the control.Radiolabeled RGD/E binding was assessed at concentrations ranging from picomolar to nanomolar after an hour at 4 °C.99m Tc cell accumulation was up to 16 times greater in the RGD group than in the RGE group.To verify the specificity, 50% of 99m Tc-labeled RGD binding to cells was inhibited by 7 nM native cyclized RGD.SE HPLC revealed that the percentage of 99m Tc-labeled RGD that bound to the isolated αVβ3 integrin protein increased as the integrin concentration increased [147].
Maraciclatide (NC100692 or diamine dioxime-Lys-Cys-Arg-Gly-Asp_Cyc-Phe-Cyspolyethylene glycol) is a cyclic peptide that contains an RGD tripeptide sequence held in a specific conformation by a disulfide moiety and one thioether bridge Cyclic RGD peptides bind to vitronectin receptors.Maraciclatide radiolabelled with technetium-99m was [ 99m Tc]Tc-RGD binding affinity was evaluated in a study using a single HUVEC cell line.αVβ3 integrins were antagonistically treated with radiolabeled RGD peptides, with RGE (Arg-Gly-Glu) serving as the control.Radiolabeled RGD/E binding was assessed at concentrations ranging from picomolar to nanomolar after an hour at 4 • C. 99m Tc cell accumulation was up to 16 times greater in the RGD group than in the RGE group.To verify the specificity, 50% of 99m Tc-labeled RGD binding to cells was inhibited by 7 nM native cyclized RGD.SE HPLC revealed that the percentage of 99m Tc-labeled RGD that bound to the isolated αVβ3 integrin protein increased as the integrin concentration increased [147].
Maraciclatide (NC100692 or diamine dioxime-Lys-Cys-Arg-Gly-Asp_Cyc-Phe-Cyspolyethylene glycol) is a cyclic peptide that contains an RGD tripeptide sequence held in a specific conformation by a disulfide moiety and one thioether bridge Cyclic RGD peptides bind to vitronectin receptors.Maraciclatide radiolabelled with technetium-99m was used as in vivo marker for vitronectin integrin receptor expression.In one study, [ 99m Tc]Tc-3PRGD2 (Figure 12) scintigraphy demonstrated increased the uptake of tracers in the joints of arthritic model rats compared to those of untreated controls.Compared with those in the controls, the expression levels of αvβ3 and CD31 were elevated in arthritic joint tissue from the rats.Bevacizumab administration ameliorated arthritis severity and reduced radiotracer uptake in the affected joints (Figure 13).This tracer has been used for identifying early synovial angiogenesis in RA patients.However, [ 99m Tc]Tc-MDP bone scanning could not differentiate between healthy controls and early-stage RA patients (Figure 14       In one study, the compound 2,3-bis(diphenylphosphino)maleic anhydride (BMA), also known as diphosphine, was combined with the cyclic peptide Arg-Gly-Asp-Dphe-Lys (RGD), and labeled with 99m Tc to specifically target the αvβ3-integrin receptor.This process led to the development of the initial diphosphine-peptide conjugate called DP-RGD.To create the radiotracer [[ 99m Tc]Tc-O 2 (DP-RGD)2]+, DP-RGD was combined with [ 99m Tc]Tc-O 4 .This radiotracer was introduced to mice induced by RA.The RA model demonstrates heterogeneity, displaying varying degrees of arthritis and symptomatic swelling among mice, and even within the joints of the same animal.Analyzing SPECT images obtained one-hour post-injection revealed a correlation between 99m Tc radioactivity accumulation and concentration in wrists and ankles (Figure 15) [150].In one study, the compound 2,3-bis(diphenylphosphino)maleic anhydride (BMA), also known as diphosphine, was combined with the cyclic peptide Arg-Gly-Asp-Dphe-Lys (RGD), and labeled with 99m Tc to specifically target the αvβ3-integrin receptor.This process led to the development of the initial diphosphine-peptide conjugate called DP-RGD.To create the radiotracer [[ 99m Tc]Tc-O2(DP-RGD)2]+, DP-RGD was combined with [ 99m Tc]Tc-O4.This radiotracer was introduced to mice induced by RA.The RA model demonstrates heterogeneity, displaying varying degrees of arthritis and symptomatic swelling among mice, and even within the joints of the same animal.Analyzing SPECT images obtained one-hour post-injection revealed a correlation between 99m Tc radioactivity accumulation and concentration in wrists and ankles (Figure 15) [150].

Matrix Metalloproteinases
Matrix metalloproteinases (MMPs) are zinc-containing, calcium-dependent endopeptidases that are involved in degrading all kinds of ECM proteins, as well as in multiple cellular processes, such as cellular adhesion, differentiation, migration, proliferation, angiogenesis, host defense, and apoptosis [167,168,169,170,171,172]. MMPs are produced by macrophages, endothelial cells, leukocytes, synoviocytes, and chondrocytes and are essential for developmental and repair processes.Inflammatory cytokines stimulate the synthesis of MMPs (Figure 19) [173,174].The most significant MMPs in RA are MMP-1, MMP-2, MMP-3, MMP-9, and MMP-13, which cleave fibrillar collagen and are correlated with VEGF and uPA.MMP-9 and MMP-13 work together to degrade joints [176,177,178,179,180].

Tumor Necrosis Factor Alpha
Tumor necrosis factor alpha (TNF-α) is a cytokine synthesized by monocytes, B cells, T cells, macrophages, and fibroblasts, which play a vital role in inflammation in RA patients [182].TNF-α regulates various functions, such as activating ECs, synovial fibroblasts, and leukocytes, inducing chemokines, cytokines, adhesion molecules, and matrix enzymes, and also stimulates neovascularization via the angiopoietin 1 and angiopoietin 2 (Ang1/Ang2) Tie2-VEGF pathways [183,184,185,186,187,188]. Barrera et al. investigated the biodistribution and susceptibility of radiolabeled adalimumab ([ 99m Tc]Tcadalimumab) in RA patients [189].The aim of this study was to determine if this antibody could target TNF and visualize the synovitis.Each patient underwent two scintigraphic imaging scans: one before administration of [99mTc]Tc-adalimumab and the other after receiving non radiolabeled anti-TNF-mAb or an intramuscular corticosteroid injection.The [ 99m Tc]Tc-TNF-mAB uptake by joints decreased when non radiolabeled TNF-mAbs were injected simultaneously.Due to its retention, TNF-α was shown to be the radiolabeled mAb target in arthritic joints.The inflammation in the group that received corticosteroids had less radiopharmaceutical uptake.The authors concluded that

Tumor Necrosis Factor Alpha
Tumor necrosis factor alpha (TNF-α) is a cytokine synthesized by monocytes, B cells, T cells, macrophages, and fibroblasts, which play a vital role in inflammation in RA patients [182].TNF-α regulates various functions, such as activating ECs, synovial fibroblasts, and leukocytes, inducing chemokines, cytokines, adhesion molecules, and matrix enzymes, and also stimulates neovascularization via the angiopoietin 1 and angiopoietin 2 (Ang1/Ang2) Tie2-VEGF pathways [183][184][185][186][187][188].Barrera et al. investigated the biodistribution and susceptibility of radiolabeled adalimumab ([ 99m Tc]Tc-adalimumab) in RA patients [189].The aim of this study was to determine if this antibody could target TNF and visualize the synovitis.Each patient underwent two scintigraphic imaging scans: one before administration of [99mTc]Tc-adalimumab and the other after receiving non radiolabeled anti-TNF-mAb or an intramuscular corticosteroid injection.The [ 99m Tc]Tc-TNF-mAB uptake by joints decreased when non radiolabeled TNF-mAbs were injected simultaneously.Due to its retention, TNF-α was shown to be the radiolabeled mAb target in arthritic joints.The inflammation in the group that received corticosteroids had less radiopharmaceutical uptake.The authors concluded that [ 99m Tc]Tc-adalimumab is an effective radiopharmaceutical for detecting clinical changes in RA disease progression (Figure 21  A study by Malviya et al. was conducted to determine which radiopharmaceutical should be utilized for treatment.[ 99m Tc]Tc-infliximab and [ 99m Tc]Tc-adalimumab were injected to assess scintigraphic images of 12 and 9 patients with active RA, respectively.Imaging scans were performed before and three months after treatment with infliximab or systemic treatment with adalimumab.There were no changes observed in the biodistribution between the two radiopharmaceuticals. Scientigraphy of [ 99m Tc]Tc-human-immunoglobulin (HIG) were also performed on two patients.Although these two patients responded clinically, cold anti-TNF-mAb treatment did not affect [ 99m Tc]Tc-HIG uptake in the joints.It was concluded that [ 99m Tc]Tc-TNF-α was superior to [ 99m Tc]Tc-HIG [190,191].
In another study, Conti et al. determined the level of TNF-mediated inflammation in the affected joints using a scintigraphic examination of a patient with RA.The patients received scintigraphic testing using 99m Tc-labeled infliximab 6 and 24 h after the injection of 555 MBq.As revealed by scintigraphy, a high level of intralesional TNF-α was indicated by the intensive build-up of [ 99m Tc]Tc-infliximab in the afflicted knee.Interestingly, there was no uptake in the inflammatory joint four months after intra-articular infliximab therapy (Figure 22) [192].A study by Malviya et al. was conducted to determine which radiopharmaceutical should be utilized for treatment.[ 99m Tc]Tc-infliximab and [ 99m Tc]Tc-adalimumab were injected to assess scintigraphic images of 12 and 9 patients with active RA, respectively.Imaging scans were performed before and three months after treatment with infliximab or systemic treatment with adalimumab.There were no changes observed in the biodistribution between the two radiopharmaceuticals. Scientigraphy of [ 99m Tc]Tc-humanimmunoglobulin (HIG) were also performed on two patients.Although these two patients responded clinically, cold anti-TNF-mAb treatment did not affect [ 99m Tc]Tc-HIG uptake in the joints.It was concluded that [ 99m Tc]Tc-TNF-α was superior to [ 99m Tc]Tc-HIG [190,191].
In another study, Conti et al. determined the level of TNF-mediated inflammation in the affected joints using a scintigraphic examination of a patient with RA.The patients received scintigraphic testing using 99m Tc-labeled infliximab 6 and 24 h after the injection of 555 MBq.As revealed by scintigraphy, a high level of intralesional TNF-α was indicated by the intensive build-up of [ 99m Tc]Tc-infliximab in the afflicted knee.Interestingly, there was no uptake in the inflammatory joint four months after intra-articular infliximab therapy (Figure 22) [192].
In one study, the certolizumab pegol (CZP), was labeled with [ 99m Tc]Tc-S-HYNIC to assess its ability to neutralize TNF-induced cytotoxicity using L929S cells.The formulation effectively blocked TNF activity at low concentrations (10 ng/mL), showing comparable effects to unlabeled CZP, with or without the HYNIC linker.Infliximab was used as a positive control, also displayed effective neutralizing activity, albeit less potent at the given doses.These findings suggest a promising radiolabeling approach for CZP, indicating potential applications in TNF-targeted imaging and therapy for inflammatory diseases such as spondyloarthritis (SpA) and RA (Figure 23) [193].In one study, the certolizumab pegol (CZP), was labeled with [ 99m Tc]Tc-S-HYNIC to assess its ability to neutralize TNF-induced cytotoxicity using L929S cells.The formulation effectively blocked TNF activity at low concentrations (10 ng/mL), showing comparable effects to unlabeled CZP, with or without the HYNIC linker.Infliximab was used as a positive control, also displayed effective neutralizing activity, albeit less potent at the given doses.These findings suggest a promising radiolabeling approach for CZP, indicat ing potential applications in TNF-targeted imaging and therapy for inflammatory diseases such as spondyloarthritis (SpA) and RA (Figure 23) [193].[193].
In another study, a radiolabeled 99m Tc, certolizumab CZP, was coupled with succin imidyl-6-hydrazino-nicotinamide.Three-time points were used to obtain whole-body im ages and images of the hands, feet, and sacroiliac joints in patients (SpA and RA).Semi quantitative scoring was applied to the immunoscintigraphic results and CZP was subse quently used to treat each patient.The scintigraphy-positive group had significantly more  In one study, the certolizumab pegol (CZP), was labeled with [ 99m Tc]Tc-S-HYNIC t assess its ability to neutralize TNF-induced cytotoxicity using L929S cells.The formulation effectively blocked TNF activity at low concentrations (10 ng/mL), showing comparabl effects to unlabeled CZP, with or without the HYNIC linker.Infliximab was used as positive control, also displayed effective neutralizing activity, albeit less potent at th given doses.These findings suggest a promising radiolabeling approach for CZP, indicat ing potential applications in TNF-targeted imaging and therapy for inflammatory disease such as spondyloarthritis (SpA) and RA (Figure 23) [193].In another study, a radiolabeled 99m Tc, certolizumab CZP, was coupled with succin imidyl-6-hydrazino-nicotinamide.Three-time points were used to obtain whole-body im ages and images of the hands, feet, and sacroiliac joints in patients (SpA and RA).Semi quantitative scoring was applied to the immunoscintigraphic results and CZP was subse quently used to treat each patient.The scintigraphy-positive group had significantly mor clinically afflicted joints or abnormal US findings in peripheral joints (p < 0.001).Bon marrow edema was found more frequently (p < 0.001) in magnetic resonance imagin (MRI) quadrants with tracer uptake in patients of SpA.At the patient level, joints with evident tracer uptake decreased probability of remaining in pain after 24 weeks o In another study, a radiolabeled 99m Tc, certolizumab CZP, was coupled with succinimidyl-6-hydrazino-nicotinamide.Three-time points were used to obtain whole-body images and images of the hands, feet, and sacroiliac joints in patients (SpA and RA).Semiquantitative scoring was applied to the immunoscintigraphic results and CZP was subsequently used to treat each patient.The scintigraphy-positive group had significantly more clinically afflicted joints or abnormal US findings in peripheral joints (p < 0.001).Bone marrow edema was found more frequently (p < 0.001) in magnetic resonance imaging (MRI) quadrants with tracer uptake in patients of SpA.At the patient level, joints with evident tracer uptake decreased probability of remaining in pain after 24 weeks of treatment with CZP (OR = 0.42, p = 0.04) compared with joints with no tracer uptake (Figure 24) [194].

Conclusions and Future Perspectives
RA is a systemic, autoimmune, inflammatory disorder that damage cartilage and joints.There has been significant advancement in the use of 99m Tc as a targeted radiopharmaceutical for molecular imaging of RA. 99m Tc SPECT/CT provides a sophisticated diagnostic method with increased accuracy due to accurate anatomical localization.
The focus on 99m Tc SPECT in hybrid imaging has emerged as a notable breakthrough in RA diagnosis.This review focused on the use of 99m Tc labeled targeting vectors for RA diagnosis, emphasizing the importance of SPECT in providing functional and molecular imaging of affected joints with RA.The short half-life and low toxicity of 99m Tc make it an ideal choice for targeted imaging of joints affected by RA.
The application of 99m Tc in RA diagnosis holds great potential for further research and clinical implementation.Future studies could confirm or explore novel ligands and targeting vectors to increase the specificity and sensitivity of 99m Tc-labeled agents, thereby improving the visualization of molecular processes related to inflammation, angiogenesis, and bone turnover in RA-affected joints.Furthermore, improvements in instrumentations, imaging technology, and the use of artificial intelligence may enhance the qualities of 99m Tc SPECT, enabling even more precise and thorough evaluations.
Collaborations between clinicians, researchers, and specialists in molecular imaging may result in the development of radiolabeled targeting vectors for personalized medicine for the accurate and customized treatment of RA, as well as for monitoring treatment responses and early disease progression assessment.

Conclusions and Future Perspectives
RA is a systemic, autoimmune, inflammatory disorder that damage cartilage and joints.There has been significant advancement in the use of 99m Tc as a targeted radiopharmaceutical for molecular imaging of RA. 99m Tc SPECT/CT provides a sophisticated diagnostic method with increased accuracy due to accurate anatomical localization.
The focus on 99m Tc SPECT in hybrid imaging has emerged as a notable breakthrough in RA diagnosis.This review focused on the use of 99m Tc labeled targeting vectors for RA diagnosis, emphasizing the importance of SPECT in providing functional and molecular imaging of affected joints with RA.The short half-life and low toxicity of 99m Tc make it an ideal choice for targeted imaging of joints affected by RA.
The application of 99m Tc in RA diagnosis holds great potential for further research and clinical implementation.Future studies could confirm or explore novel ligands and targeting vectors to increase the specificity and sensitivity of 99m Tc-labeled agents, thereby improving the visualization of molecular processes related to inflammation, angiogenesis, and bone turnover in RA-affected joints.Furthermore, improvements in instrumentations, imaging technology, and the use of artificial intelligence may enhance the qualities of 99m Tc SPECT, enabling even more precise and thorough evaluations.
Collaborations between clinicians, researchers, and specialists in molecular imaging may result in the development of radiolabeled targeting vectors for personalized medicine for the accurate and customized treatment of RA, as well as for monitoring treatment responses and early disease progression assessment.
In summary, using 99m Tc labeled targeting vectors unveils new paths for innovative strategies in diagnosis as well as understanding the complexities of RA. 99m Tc applications

Figure 1 .
Figure 1.Normal and rheumatoid arthritis joints.Reprinted from open access source (CC BY) [13

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Figure 9 .
Figure 9. MMR expression in arthritic joints can be detected via in vivo imaging using MMR-specific nanobodies.Micro-CT scans were followed by SPECT imaging.(A) Mice that were immunized but did not exhibit clinical arthritis symptoms were injected with labeled MMR nanobodies or BCII10 control nanobodies.(B) Mice injected with labeled MMR nanobodies or BCII10 control nanobodies were symptomatic of arthritis in both hind limbs.Reprinted from open access source (CC BY 4.0) [53].

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Figure 10 .
Figure 10.[ 99m Tc]Tc scVEGF SPECT of sterile right thigh abscess.Reprinted from open access source (CC BY 4.0) [130].Galli et al. used a human VEGF165 analog radiolabeled with 99m Tc to assess VEGFR expression in HUVECs and in a mouse model (ARO, HT29, and K1).In vitro, [ 99m Tc]Tc-VEGF was shown to bind to HUVECs, and in vivo, [ 99m Tc]Tc-VEGF was shown to bind to xenograft tumors in mice (ARO, K1, and HT29).Comparison of in vivo data with immunohistochemical analysis of excised tumors revealed an inverse correlation between [ 99m Tc]Tc-VEGF165 uptake and histologically detected VEGF [131].

Figure 10 .
Figure 10.[ 99m Tc]Tc scVEGF SPECT of sterile right thigh abscess.Reprinted from open access source (CC BY 4.0) [130].Galli et al. used a human VEGF165 analog radiolabeled with 99m Tc to assess VEGFR expression in HUVECs and in a mouse model (ARO, HT29, and K1).In vitro, [ 99m Tc]Tc-VEGF was shown to bind to HUVECs, and in vivo, [ 99m Tc]Tc-VEGF was shown to bind to xenograft tumors in mice (ARO, K1, and HT29).Comparison of in vivo data with immunohistochemical analysis of excised tumors revealed an inverse correlation between [ 99m Tc]Tc-VEGF165 uptake and histologically detected VEGF [131].

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Figure 11 .
Figure 11.The pathway by which RGD-containing fibronectin fragments induce cartilage damage and proteoglycan loss.Reprinted from open-access source (CC BY 4.0) [142].

Figure 11 .
Figure 11.The pathway by which RGD-containing fibronectin fragments induce cartilage damage and proteoglycan loss.Reprinted from open-access source (CC BY 4.0) [142].

Figure 20 .
Figure 20.Investigation of the in vivo distribution and excretion of barbiturates following intravenous administration in adult C57BL/6 mice.(A) Biodistribution of the radiotracers [ 99m Tc]Tc-MEA39, [ 99m Tc]Tc-MEA61, and [ 99m Tc]Tc-MEA223.(B) The left ventricular volume of interest (VOI) was used to calculate dynamic in vivo blood radioactivity.(C) Hepatic accumulation of the radiotracer in vivo.(D) The relative proportion of the hepatobiliary and renal elimination pathways, as well as the radioactivity accumulation (shown as a percentage of the injected dosage), in the excretion organs (liver, gallbladder, intestine, kidney, and bladder) 10 and 90 min post-injection.The mean ± SD (n = 4-5) is displayed along with the ID (injected dose) and left image orientation (L).Statistical significance was determined via two-way ANOVA and Tukey's post hoc test.Stars denote the significance of variations in [ 99m Tc]Tc-MEA223′s radiotracer uptake relative to that of the other compounds: *** p < 0.05, ** p < 0.01, *** p < 0.001.Reprinted from open access source [181].

Figure 20 .
Figure 20.Investigation of the in vivo distribution and excretion of barbiturates following intravenous administration in adult C57BL/6 mice.(A) Biodistribution of the radiotracers [ 99m Tc]Tc-MEA39, [ 99m Tc]Tc-MEA61, and [ 99m Tc]Tc-MEA223.(B) The left ventricular volume of interest (VOI) was used to calculate dynamic in vivo blood radioactivity.(C) Hepatic accumulation of the radiotracer in vivo.(D) The relative proportion of the hepatobiliary and renal elimination pathways, as well as the radioactivity accumulation (shown as a percentage of the injected dosage), in the excretion organs (liver, gallbladder, intestine, kidney, and bladder) 10 and 90 min post-injection.The mean ± SD (n = 4-5) is displayed along with the ID (injected dose) and left image orientation (L).Statistical significance was determined via two-way ANOVA and Tukey's post hoc test.Stars denote the significance of variations in [ 99m Tc]Tc-MEA223 ′ s radiotracer uptake relative to that of the other compounds: * p < 0.05, ** p < 0.01, *** p < 0.001.Reprinted from open access source [181].

Figure 21 .
Figure 21.Scintigraphic images of an active RA patient's hands and entire body after receiving an injection of [ 99m Tc]Tc-human-anti-TNF-mAb. (A) The initial imaging studies.(B) Following the injection of an excess of unlabeled anti-TNF mAb, there was a decrease in uptake in the joints (hands detail), but there was no change in uptake in the reticuloendothelial organs (whole body).Reprinted from open access source [189].

Figure 21 .
Figure 21.Scintigraphic images of an active RA patient's hands and entire body after receiving an injection of [ 99m Tc]Tc-human-anti-TNF-mAb. (A) The initial imaging studies.(B) Following the injection of an excess of unlabeled anti-TNF mAb, there was a decrease in uptake in the joints (hands detail), but there was no change in uptake in the reticuloendothelial organs (whole body).Reprinted from open access source [189].

Figure 24 .
Figure 24.Certolizumab pegol labeled with Tc99m was distributed in the hands, feet, and sacroiliac joints 4-5 h after injection.(A) A patient with active rheumatoid arthritis exhibited a polyarticular pattern of joints without involvement of distal interphalangeal joint (DIP).(B) Left second digit distal interphalangeal joint uptake in polyarticular psoriatic arthritis patients.(C) Tracer uptake in dactylitis patient of the fourth digit.(D) SPECT-CT scan of a patient's right foot showing Achilles tendon enthesitis and spondyloarthritis.(E) Fusion of sacroiliac joint SPECT and MRI images in an axial spondyloarthritis patient.Reprinted from open access source (CC BY-NC-ND 4.0) [194].

Figure 24 .
Figure 24.Certolizumab pegol labeled with Tc99m was distributed in the hands, feet, and sacroiliac joints 4-5 h after injection.(A) A patient with active rheumatoid arthritis exhibited a polyarticular pattern of joints without involvement of distal interphalangeal joint (DIP).(B) Left second digit distal interphalangeal joint uptake in polyarticular psoriatic arthritis patients.(C) Tracer uptake in dactylitis patient of the fourth digit.(D) SPECT-CT scan of a patient's right foot showing Achilles tendon enthesitis and spondyloarthritis.(E) Fusion of sacroiliac joint SPECT and MRI images in an axial spondyloarthritis patient.Reprinted from open access source (CC BY-NC-ND 4.0) [194].